Discovery and verification of mmu_Circ_26986/hsa_Circ_0072463 as a potential biomarker and intervention target for sepsis-associated acute kidney injury

Approximately 60% of septic patients developed acute kidney injury (AKI). The mortality rate of septic AKI (SA-AKI) is two to three times higher than that of septic without AKI (SA-non-AKI). The actual functions and mechanisms of CircRNAs in the pathophysiology of SA-AKI remain incompletely understood. Herein, we observed that the mmu_Circ_26986 could be induced by lipopolysaccharide (LPS) and cecum ligation and puncture (CLP) in BUMPT cell line and C57BL/6 mouse kidney, respectively. Functionally, mmu_Circ_26986 suppressed BUMPT cell apoptosis induced by LPS. Mechanistically, mmu_Circ_26986 sponged miRNA-29b-1-5p to upregulate the expression of PAK7. Overexpression of mmu_Circ_26986 ameliorated the progression of CLP-stimulated AKI through miRNA-29b-1-5p/PAK7 axis. In addition, we found that hsa_Circ_0072463, homologous to mmu_Circ_26986, suppressed LPS-induced HK-2 cells apoptosis via regulation of miRNA-29b-1-5p/PAK7 axis. Furthermore, sepsis patients with AKI had a higher level of hsa_Circ_0072463 compared to those without AKI. The sensitivity, specificity and AUC of hsa_Circ_0072463 were 78.8%, 87.9% and 0.866, respectively. Spearman's test indicated a noticeable positive correlation between plasma hsa_Circ_0072463 and serum creatinine in sepsis patients (r = 0.725). In summary, this study reveals that the mmu_Circ_26986/hsa_Circ_0072463 miRNA-29b-1-5p/PAK7 axis mediates septic AKI, and hsa_Circ_0072463 is a potential diagnostic marker for septic AKI. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-023-05079-x.


Introduction
Approximately 60% of patients with sepsis suffer from sepsis-associated acute kidney injury (SA-AKI), exacerbating sepsis morbidity and mortality [1].Therefore, it is crucial to early identify SA-AKI and investigate its underlying mechanism [2].Recent data have identified more than ten proteins, such as TIMP2 and IGFBP7, with potential diagnosis values for SA-AKI [3,4].Additionally, three underlying mechanisms, including microcirculatory dysfunction [5], inflammation [6], and metabolic reprogramming [7], contribute to the progression of SA-AKI.However, there is currently no effective treatment method other than kidney replacement therapy to prevent the progression of SA-AKI [8][9][10].Hence, our goal is not only to explore the pathophysiology of SA-AKI, but also to identify better early biomarkers for it.The significant aspect is that the new molecule serves as both an intervention target and a potential biomarker for SA-AKI.
Circular RNAs (CircRNAs), a specific class of RNA molecules, belong to non-coding RNAs (ncRNAs) [11].In comparison with linear RNAs, CircRNAs exhibit greater stability, possess a longer lifespan, and demonstrate resistance to RNase R, making them ideal biomarkers for human diseases [12,13].Moreover, CircRNAs are responsible for the progression of various diseases and have become therapeutic targets [14].Recent research has identified several CircRNAs responsible for the progression of SA-AKI [15].Only one study has indicated that Circ_0020339 promotes the development of SA-AKI and acts as a diagnostic marker [16].In this study, we focus on mmu_Circ_26986, located in Arl15, whose role and diagnostic value in SA-AKI remain largely unclear.
Our study indicated that lipopolysaccharide (LPS) and cecum ligation and puncture (CLP) could induce the expression of mmu_Circ_26986 both in cell and animal experiments.Mmu_Circ_26986 suppressed LPS-stimulated apoptosis in BUMPT cells by targeting the miRNA-29b-1-5p/PAK7 axis.CLP-induced SA-AKI in mice was ameliorated by the overexpression of mmu_Circ_26986.Finally, we found that hsa_Circ_0072463, homologous to mmu_Circ_26986, serves as an early diagnostic marker for SA-AKI.

Animal models
Circ_26986 or control expression vectors (25 μg DNA/injection) were administered through the tail vein of C57BL/6 J male mice (8-10 weeks old), and then subjected to CLP, saline injection as a sham operation as a control, respectively.Kidney tissues were acquired for assessing renal morphology and function at 18 h after CLP treatment.The study involved animal testing in adherence to the guidelines set by the Institutional Committee for the Care and Use of Laboratory Animals at the Second Xiangya Hospital.Throughout the experiments, the animals were provided unrestricted access to standard water and food and followed a 12-h light-dark cycle.

Cecum ligation and puncture model
C57 mice weighing 21-25 g were selected and fasted for 12 h before surgery.After anaesthesia was administered by intraperitoneal injection according to body weight, the animals were fixed supine on a surgical plate, the abdominal surgical regions were routinely disinfected and dehairing was performed, and a 2-cm-long incision was made on the abdominal wall with a scalpel in aseptic conditions.An 18-gauge needle was used to perforate the ligated end and a small amount of faeces was squeezed out, avoiding damage to the blood vessels as much as possible, and then the skin and peritoneum were closed intermittently with a 4-gauge silk suture, while 50 ml/kg saline was injected for antishock.

qRT-PCR analysis
Trizol Reagent (Invitrogen) was utilized for total RNA extraction from BUMPT cells, HK-2 cells, and kidneys of C57BL/6 J mice.Next, approximately 40 ng of total RNA was reverse transcribed to cDNA using the Prime-Script-RT-Reagent-Kit and the gDNA-Eraser-Kit (RR047A; TaKaRa, Japan).qRT-PCR and SYBR Green (AG11728; Accurate Biotechnology (HUNAN) CO., LTD, ChangSha China) were employed to detect the expression levels of circRNA, micoRNA, and target gene mRNA.Relative quantification was performed by Roche LC 480 determination of ΔCt values (F.Hoffmann-La Roche, Ltd.).

FISH analysis
BUMPT cell line or kidney tissues were fixed with paraformaldehyde for 5 min and then permeabilized with prehybridization solution and hybridization solution.Mmu_Circ_26986 fluorescent probe and miRNA-29b-1-5p fluorescent probe (Ruibo, China) were hybridized overnight at 37°.On the next day, nuclei were blocked and stained with DAPI, followed by fluorescence imaging analysis using a laser scanning confocal microscope.U6 nuclear positivity and 18S rRNA cytoplasmic positivity were used for references.

Flow cytometry assessment
Annexin V-FITC/PI staining was performed to determine apoptosis.Briefly, BUMPT or HK-2 cell lines were harvested and resuspended with binding buffer, and subsequently stained with annexin V-FITC and PI.After incubation for 15 min in the dark, 200 uL of binding buffer was added and examined using a Northern Light flow meter (Cytek Biosciences).Apoptosis rate was the percentage of late apoptotic (AnnexinV + /PI +) and early apoptotic (AnnexinV + /PI-) cells to cells.

Renal function, morphology, and TUNEL staining
The concentrations of BUN and creatinine were detected (Nanjing Jianjian Bioengineering Research Institute, China).H&E staining was used to evaluate the degree of renal tubular injury: renal tubules were markedly dilated and cellular flattening was scored as 1 point; brush border injury was scored as 1 point, detachment was scored as 2 points; titularity was scored as 2 points; and the presence of detached, necrotic cells in the lumen of renal tubules (which did not become tubular or cellular debris) counted as 1 point.TUNEL staining was applied to determine renal cell apoptosis, positively stained cells were counted and stained samples were evaluated using Zeiss microscope-equipped software.

Human samples
The definition and grading of SA-AKI were referred to the consensus guidelines and KDIGO criteria, respectively.Human blood was collected from healthy volunteers (n = 33), SA-non-AKI patients (n = 33), and SA-AKI patients (n = 33), and then centrifuged to extract plasma or serum, followed by storage at -80°.The research methodology received approval from the Second Review Committee of Xiangya Hospital, People's Republic of China.Prior to participation, all individuals provided informed consent for their involvement in the study.

Statistical analysis
GraphPad Prism 9.4 was utilized for statistical analyses.All values are shown as mean ± SD.Differences between two or more groups were examined with Student's T-test or one-way ANOVA followed by Tukey's post-hoc test.ROC curve analysis was also performed using GraphPad.The

Mmu_Circ_26986 expression is elevated in LPS-induced BUMPT cells and CLP-induced mice AKI
We examined the expression of mmu_Circ_26986 in BUMPT cell line and C57BL/6 mouse model induced by LPS and CLP, respectively.Firstly, qRT-PCR data unveiled that mmu_Circ_26986 was highly expressed at 6 h, gradually increased at 12 h, and peaked at 24 h (Fig. 1A).Secondly, to determine the in vivo expression of mmu_Circ_26986, a sepsis animal model was constructed by CLP in C57BL/6 J mice.Renal function detection indicated a slight increase in BUN and Creatinine induced by CLP at 6 h, reaching a peak at 18 h (Fig. 1B, C).H&E and TUNEL staining verified that CLP gradually exacerbated renal tubular injury and apoptosis at 6 h and 18 h (Fig. 1D-G).The qRT-PCR data also showed that mmu_Circ_26986 was highly expressed at 9 h and peaked at 18 h (Fig. 1H).Correlation analysis Fig. 2  revealed a high correlation between the mmu_Circ_26986fold change (R = 0.957) and the renal cell apoptosis rate (Fig. 1I).FISH analysis demonstrated that mmu_Circ_26986 mainly localized in the cytoplasm of BUMPT and mouse kidney tubular cell lines (Fig. 1J).These findings imply that mmu_Circ_26986 is related to the progression of SA-AKI.

Downregulation of mmu_Circ_26986 enhances LPS-stimulated BUMPT cell apoptosis
To determine the functions of mmu_Circ_26986 in BUMPT cell apoptosis after LPS treatment, mmu_Circ_26986 with siRNA was used.qRT-PCR analysis indicated that mmu_ Circ_26986 siRNA suppressed its expression levels under LPS and basal conditions (Fig. 2A).Flow cytometry analysis demonstrated that mmu_Circ_26986 siRNA exaggerated LPS-stimulated BUMPT cell death (Fig. 2B&C), and this effect was verified by the immunoblotting results of cleaved-caspase3 (CC3) (Fig. 2D&E).These results suggest that mmu_Circ_26986 has an anti-apoptotic function during LPS treatment.

Mmu_Circ_26986 overexpression ameliorates LPS-stimulated BUMPT cell apoptosis
To further support the above findings, the mmu_Circ_26986 plasmid was applied.qRT-PCR data indicated that overexpression of mmu_Circ_26986 upregulated its expression under LPS and basal conditions (Fig. 3A).Flow cytometry analysis showed that overexpression of mmu_ Circ_26986 attenuated LPS-stimulated BUMPT cell death (Fig. 3B&C), and this effect was further verified by the
We further elucidated the mechanism underlying the antiapoptotic action of PAK7 in BUMPT cell line.Immunoblot analysis showed that PAK7 siRNA reduced the expression of wnt and β-catenin, but promoted the expression of pβ-catenin (Supplement Fig. 1).These results demonstrate that PAK7 may play an anti-apoptotic role through the wnt/ β-catenin pathway.

Overexpression of hsa_Circ_0072463 inhibits LPS-stimulated HK-2 cell apoptosis
To further explore whether mmu_Circ_26986 has a homology with human CircRNA, the Circbase database was used.We found that mmu_Circ_26986 with homology hsa_ Circ_0072463 was the most likely target (Fig. 9A).We further investigated the expression of hsa_Circ_0072463 upon LPS exposure.The qRT-PCR results demonstrated that hsa_ Circ_0072463 was overexpressed at 6 h and peaked at 24 h (Fig. 9B).To validate the function of hsa_Circ_0072463, hsa_Circ_0072463 plasmid was transfected into HK-2 cell Fig. 9 Overexpression of hsa_Circ_0072463 attenuates LPSstimulated HK-2 cell apoptosis.A Sequence comparison of mmu_ Circ_26986 and hsa_Circ_0072463.Sequence conservativeness was examined using the blast function of CircBase webtool (http:// circr na.org/ cgi-bin/ webBl at).According to the blast result, when the homology > 80%, the higher the score, the higher the homology of hsa_ Circ_RNA is considered.HK-2 cells were exposed to LPS (50 μg/ mL) for 6, 12 or 24 h.B qRT-PCR assessment of hsa_Circ_0072463 levels in cells.HK-2 cell line was transfected with hsa_Circ_0072463 plasmid and then were subjected to LPS 24 h.qRT-PCR was utilized for examining hsa_Circ_0072463 (C) and hsa-miRNA-29b-1-5p (D) levels.E Flow cytometry evaluation of HK-2 cell apoptosis.F Calculation of apoptosis rate.G Immunoblot assessment of the expression of C3, CC3, and PAK7 in HK-2 cells.H Grayscale evaluation of the immunoblot bands of C3, CC3, and PAK7 in HK-2 cells.Mean ± SD (n = 6).#p < .05 vs. SC + saline group.*p < .05 vs. SC + I/R group ◂ line and subsequently exposed to LPS.The qRT-PCR data revealed that overexpression of hsa_Circ_0072463 increased its expression but reduced miRNA-29b-1-5p expression under the basal and LPS conditions (Fig. 9C, D).The flow cytometry analysis results showed that hsa_Circ_0072463 overexpression noticeably ameliorated LPS-stimulated HK-2 cell death (Fig. 9E, F), which was validated by the immunoblot analysis of CC3 protein downregulation and PAX7 upregulation (Fig. 9G, H).These findings imply that hsa_Circ_0072463 shares the same function and mechanism with mmu_Circ_26986 during LPS stimulation.

Homologous hsa_Circ_0072463 of mmu_Circ_26986 is a biomarker of SA-AKI
To evaluate whether plasma hsa_Circ_0072463 could act as a diagnostic marker in patients with SA-AKI, we collected the plasma specimens from SA-AKI patients (n = 33), non-AKI patients (n = 33), and age-matched healthy subjects (n = 33), and the expression of hsa_Circ_0072463 was then analyzed.Absolute RT-PCR evaluation indicated that the expression of hsa_Circ_0072463 was induced in septic non-AKI compared with controls, and its expression was higher in sepsis patients than in septic non-AKI patients (Fig. 10A).
The clinical features of SA-AKI and non-SA-AKI patients as well as age-matched healthy subjects (n = 99) are shown in Supplement Table 1.We further assessed the diagnostic potential of hsa_Circ_0072463 by generating a subject work characteristics (ROC) curve.The sensitivity, specificity and AUC of plasma hsa_Circ_0072463 were 78.8%, 87.9% and 0.866 (95% confidence interval 0.775-0.957),respectively (Fig. 10B).Interestingly, the expression level of plasma hsa_Circ_0072463 in KIDGO stage2 was higher compared to KIDGO stage1.However, its expression level in KIDGO stage3 was not significantly increased compared to KIDGO stage2 (Fig. 10C).Spearman's correlation coefficient showed the positive correlation between plasma hsa_Circ_0072463 levels and serum creatinine (Fig. 10D).In summary, hsa_ Circ_0072463 may function as an early diagnostic marker for SA-AKI.
A recent study highlighted circ_0020339 as an early diagnostic marker for SA-AKI, but the methodology lacked sensitivity and specificity calculations [16].In our research, we identified, for the first time, a homologue of mmu_Circ_26986, hsa_Circ_0072463, which mediates LPS-stimulated HK-2 cell apoptosis through the regulation of the miRNA-29b-1-5p/PAK7 axis.Furthermore, we validated hsa_Circ_0072463 as an early diagnostic marker for SA-AKI, as supported by its higher sensitivity of 78.79%, higher specificity of 87.88%, and a stronger correlation with serum creatinine (a conventional diagnostic marker of kidney injury) (Fig. 10).However, it is important to note that our study is limited by the sample size, and future research should expand the cohort to further explore the diagnostic potential of hsa_Circ_0072463 for SA-AKI.
In conclusion, our data reveal that mmu_Circ_26986/ hsa_Circ_0072463 exerts an anti-apoptotic function by targeting the miRNA-29b-1-5p/PAK7 axis.These CircR-NAs hold promise as therapeutic targets for SA-AKI, with hsa_Circ_0072463 emerging as a reliable biomarker for the condition (see Fig. 11).

Supplementary Information
The online version contains supplementary material available at https:// doi.org/ 10. 1007/ s00018-023-05079-x.Fig. 11 Modelling and mechanism of mmu_Circ_26986/hsa_ Circ_0072463 in septic AKI.In sepsis AKI, the expression of mmu_Circ_26986 is increased.mmu_Circ_26986 competitively acts on miRNA-29b-1-5p, increases PAK7 expression, and exhibits anti-apoptotic effects on cell and animal models.Homologous hsa_ Circ_0072463 is the most likely target of mmu_Circ_26986.Hsa_Circ_0072463 may serve as an early diagnostic marker and a new therapeutic target for septic AKI

Fig. 1
Fig.1 Effect of LPS on Circ_26986 expression in BUMPT cell and CLP mouse models.BUMPT cell line was exposed to LPS (300 μg/mL) for 6, 12 or 24 h.A qRT-PCR detection of Circ_26986 expression in cells.C56BL/6 mice were exposed to CLP for 9 and 18 h.Time-dependent increases of BUN (B) and serum creatinine (C) in CLP-induced sepsis mice.D, E H&E and TUNEL staining for assessing the degree of impairment.Score bar: 50 µm.F H&E damage score.G TUNEL positive cells/mm 2 .H qRT-PCR was applied to determine Circ_26986 expression.I Correlation coefficients of Circ_26986 expression in kidney tissues with CLP-stimulated apoptosis.J RNA-FISH assay of Circ_26986 localization in BUMPT cell line and mouse kidney tissues.Nuclear and cytoplasmic labeling were performed with U6 and 18S as the controls, respectively.Finally, Circ_26986 was also localized in the kidney via RNA-FISH assay.Score bar: 10 µm.#p < .05,LPS 12 and 24 h or CLP 9 h and 18 h groups compared with Control or Sham groups